Membrane proteins account for -~3O% of proteins encoded by various genomes and play critical roles in biological organisms. However, structural biology of membrane proteins lags far behind that of their soluble counterparts, which hampers efforts to understand their mechanisms and to fully exploit them as drug targets. The bacterial chemoreceptor family is an ideal system for investigating the molecular mechanism of transmembrane signaling, a fundamental process mediated by membrane proteins. During the current funding period, we have established a site-directed solid-state NMR distance measurement approach capable of measuring local structure in large membrane proteins, and have made the first distance measurements in the intact, membrane-bound Ser receptor with sufficient resolution to measure the subtle changes thought to transmit the signal. [unreadable] [unreadable] Aims 1-3 of the this proposal will use site-directed solid-state NMR to measure interhelical distances in the periplasmic, transmembrane, and cytoplasmic domains of the intact, membrane-bound Ser receptor to map the intra- and inter-subunit conformational changes induced by ligand-binding and receptor methylation. These experiments will also refine the structural model of the transmembrane helices, measure the secondary structure of the critical linker region (Aim4), test proposed dimer-dimer contacts in the cytoplasmic domain to elucidate the structure of receptor clusters, and measure the orientation of amides throughout the receptor to test the overall structural model (Aim 5). [unreadable] [unreadable] The overall goal is to develop and use an integrated solid-state NMR/biochemical approach to obtain high-resolution information on the intact, membrane-bound Ser receptor, which is unavailable by other methods, to reveal the structural basis of the mechanism of transmembrane signaling. These integrated approaches will be applicable to studies of structure & function in other important membrane protein systems.